Distinct normal cells-of-origin may underlie the phenotypic heterogeneity of human breast carcinomas. Perturbations of the normal epigenetic programs of various cells-of-origin may play roles in tumor initiation, metastatic progression, and resistance to therapies. To begin investigating these issues, we characterized. the comprehensive DMA methylation and gene expression profiles of differentiated CD24+ luminal epithelial and CD44+ stem cell-like cells from normal and neoplastic human breast tissue. We identified cell-typespecific DMA methylation patterns that correlated strongly with the histopathological subtypes and clinical outcome of human breast carcinomas. The relatedness of normal and neoplastic DNA methylation patterns was dependent on breast tumor subtypes. Based on these observations, we hypothesize that genes associated with distinct chromatin patterns in mammary epithelial stem and differentiated cells play key roles in determining stem-cell function and lineage-specific differentiation, and that perturbation of these patterns alters cell fate. Furthermore, we hypothesize that normal cell type-specific chromatin patterns are perturbed in breast tumors and that they vary depending on breast tumor types. We propose three specific aims to test these hypotheses. Aim 1: To characterize the histone methylation profiles of human mammary epithelial stem and differentiated cells isolated from normal human breast tissue. We will use ChIP (chromatin immunoprecipitation) and quantitative PCR followed by ChlPSeq genome-wide studies. Aim 2: To characterize the histone methylation profiles of stem cell-like and more differentiated breast cancer cells isolated from different breast tumor subtypes. We will analyze the histone methylation of stem cell-like and more differentiated cells-from different stage tumors of several subtypes (e.g., luminal, HER2+, and basallike tumors) as described in Aim 1. Aim 3. To determine the functional relevance of the cell type-specific epigenetic patterns. To determine the functional relevance of cell type-specific histone methylation patterns, we will analyze the consequences of perturbing these patterns on cell fate and differentiation both in normal and neoplastic human breast tissue.